Plant hormones cytokinins (CKs) play a crucial role in various physiological processes such as plant development, stress or senescence. Their levels are very precisely regulated in various tissues or subcellular compartments. The mechanism of CKs regulation is mediated by proteins participating in the CKs biosynthesis and activation, degradation, transport or perception. Cytokinin dehydrogenases (CKX, EC 1.5.99.12) are the enzymes that inactivate CKs hormones by irreversible degradation of their molecule. There are several isoforms of CKX in plants, wherein each isoform has a different specificity for the cytokinins (Bilyeu et al. 2001, Plant Phys. 125:378; {hacek over (S)}mehilová et al. 2009, J. Exper. Bot. 60:2701). A study of transcript levels of CKX isoforms in the leaves of Arabidopsis, maize and barley revealed different expression patterns among the isoforms during leaf senescence and stress, accounting for a confirmed decrease of cytokinins. On the contrary, it was shown that the CK metabolism is accelerated during salt and osmotic stress as evidenced by a moderate increase of CK active forms (Vyroubalová et al. 2009, Plant Physiol. 151:433). Taken together, these observations suggest distinct roles of different CKs forms during various physiological processes such as abiotic stress (Kuiper et al. 1990, Plant and Soil 123, 243; Davies & Zhang 1991, Ann. Rev. Plant Physiol. Plant Mol. Biol. 42, 55; Havlová 2008, Plant Cell Env. 31:341) or senescence (Eisinger 1977, Plant Phys. 90, 1316; Lara et al. 2004, Plant Cell 16:1276). However, the exact role of the cytokinins in the mentioned processes, especially during leaf senescence, has yet to be elucidated. Leaf senescence is a key step in the life cycle of annual plants, playing an indispensable role in plant reproduction, productivity, or plant survival by means of stress handling. The senescence is the last step of leaf development occurring in an age-dependent manner, which was shown to be a complex and highly regulated process. During this process, in a senescing leaf, the stored nutrients are re-mobilized and the following degradative processes take place leading to sustaining of younger, fully developing organs such as young leaves, fruits or grains. One of the conspicuous visual symptoms of leaf senescence is the loss of chlorophyll pigments (yellowing), which is caused by replacing the chloroplast content by the products of cell degradation processes. The leaf yellowing is further followed by desiccation and eventual abscission. On the cellular or molecular level, the processes contributing to these visual symptoms include, in addition to chloroplast disintegration, also loss of proteins and nucleic acids of the cell. Molecular fundamentals of these events are under the control of very varied plant regulatory mechanisms. Although the senescence occurs in an age-dependent manner in many species, its initiation and progression can be modulated by a variety of environmental factors such as temperature, mineral deficiency, and drought conditions, as well as by internal factors such as plant growth regulators. The leaf senescence is generally balanced by a range of phytohormones, wherein the cytokinins decrease is correlated with senescing processes in the leaves (Winkler et al. 2006, J. Exper. Bot. 57:391). Moreover, it was shown that exogenous application of cytokinins inhibits the degradation of chlorophyll and proteins of the photosynthetic apparatus (He et al 2005, J. Exper. Bot. 56, 1117-1128). The strategy leading to delayed senescence involved the transgenic expression of isopentenyltransferase (IPT; 2.5.1.27), an enzyme of the cytokinin biosynthetic pathway. The transgenic plants with elevated cytokinin levels showed delayed senescence (Gan et al. 1995, Science 270:1986). A different approach to maintaining sufficient levels of active cytokinins within plant tissues employs the inhibition of cytokinin dehydrogenase. However, the cytokinin-deficient transgenic plants did not show any earlier onset of leaf senescence (Werner et al. 2003, Plant Biol. 8:371). Consequently, it is not proven yet whether the decrease in the cytokinin levels acts as a trigger of senescence or whether it is only a senescence accompanying phenomenon. Furthermore, numerous enzymes implicated in providing essential activities for the initiation and progression of the senescence include proteases, nucleases, and other degradative enzymes, as well as enzymes involved in chloroplast dismantling and chlorophyll breakdown, which were indicated to be co-induced by cytokinins. The nutrients re-mobilization and the sustenance of other plant parts are pursued via source-sink relation mediated by the enzymes of the apoplastic phloem unloading pathway. One of the hexoses-regulating enzymes, an extracellular invertase, was observed to be co-induced by cytokinins. However, it was further demonstrated that the extracellular invertase is required for the delaying of senescence, thus indicating the extracellular invertase as a key underlying mechanism of the delaying of senescence by cytokinins (Lara et al. 2004, Plant Cell 16:1276). Involvement of cytokinin dehydrogenase activity in the process of senescence was expected, since distinct isoforms of these cytokinin deactivating enzymes were experimentally proved to be up-regulated on the gene expression level and in enzymatic activity measurements ({hacek over (S)}romová Lucie, diploma thesis 2006, P{hacek over (r)}F UP).
The fact that cytokinins can delay senescence is known since kinetin (N6-furfurylaminopurine) was first isolated (Miller et al. 1956, J. Am. Chem. Soc. 78:1375) and tested for its anti-senescence properties on detached Xanthium leaves (Richmond & Lang 1957, Science 125:650). A variety of cytokinin derivatives were observed to delay senescence in various plant species (Zhang & Letham 1989, J. Plant Growth Regul. 8:181; Kuhnle et al. 1977, Physiol. Plant. 41:14; Eisinger 1977, Plant Physiol. 59:707; Kudryakova et al. 2001, Plant Growth Regul. 56:21). Some kinetin derivatives are already known (Zhang & Lethmm 1989, J. Plant Growth Regul. 8:181; Fox et al. 1971, Plant Phys. 47:275). We have synthesized several novel derivatives substituted at the N9-position of purine with short 9-halogenalkyls and determined their anti-senescence and anti-ageing properties in different bioassays.
In an optimum concentration, the cytokinins are compounds with a stimulatory activity on cell proliferation; however, their effect is rather inhibitory in the concentrations exceeding 10 μM (Holub et al., 1998, Plant Growth Regul, 26, 109-115). It is also generally known that the cytokinin molecule acts as a negative regulator on root elongation and lateral root branching (Werner et al., 2001, Proc Natl Acad Sci USA. 98, 10487-92; Werner et al., 2003, Plant Cell, 15, 2532-50). The exogenous application of cytokinins in the concentrations exceeding 1 μM usually results in a total suppression of root growth. The aim of this invention is the preparation of novel cytokinin N9-substituted derivatives, which have unique activities in the regulation of plant growth and are not toxic to plant and animal cells at the same time.
Purine derivatives substituted in position 6 by benzyl, phenyl, furyl, furfuryl and naftyl groups (optionally further substituted), and in position 9 by lower alkyl groups, optionally containing —O— or —S— groups, fall within the general formula of GB 1,027,756 (1966) for use to promote growth and/or prevent decomposition processes in plants, but in the examples of this invention only compounds containing tetrahydropyranyl group at 9 position are disclosed, and thus the activity of compounds with lower alkyl groups in position 9 is not shown and with regard to the wide range of compounds falling into the general formula it cannot be concluded that these compounds would have the same activity. Furthermore, similar derivatives containing an optionally substituted phenyl or naftyl groups in position 6 and C1-C6 alkyl or phenyl, optionally substituted phenyl, in position 9, for regulation of plant growth fall within the general formula of EP 0155911. The activity of these derivatives is however not determined in this document; only the activity of derivatives with mesithyl substituents in position 9 is shown. With regard to the generality of the general formula and the structural differences between mesithyl and alkyl substitutents it is impossible to make any conclusions regarding the real activity of 9-alkyl substituents. Ramzayeva et al., published in {hacek over (Z)}urnal organi{hacek over (c)}eskoj chimii (1988, 1090-1094) describes the procedure for substitution of 6-substituted purines by halogenalkyls. The compounds disclosed in this publication contain benzylamino or methylthio groups in position 6 and 2-chloroethyl, 2-bromoethyl and 3-chloropropyl group in position 9, respectively. Nevertheless, this paper is not mentioning any use of the above mentioned compounds.